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Team:OUC-China/Project/PooSb/pc
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<h2>The promoting cycle constituted by five devices</h2> | <h2>The promoting cycle constituted by five devices</h2> | ||
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<h2>he final results we want to achieve</h2> | <h2>he final results we want to achieve</h2> | ||
<p> When and only when the five kinds of bacteria were cultured together in complementary medium without leucine, they will survive and showing five different fluorescent proteins when detected by light with different wave lengths. Once any kind of bacteria is dead or lacked in the culture, the whole cycle will break off as a result of the AHLs ‘ signal blocking . correspondingly, AHL synthetase, LeuB and reporter protein’s expression will decreased gradually. Finally, this system will end up with a dead end.<br> | <p> When and only when the five kinds of bacteria were cultured together in complementary medium without leucine, they will survive and showing five different fluorescent proteins when detected by light with different wave lengths. Once any kind of bacteria is dead or lacked in the culture, the whole cycle will break off as a result of the AHLs ‘ signal blocking . correspondingly, AHL synthetase, LeuB and reporter protein’s expression will decreased gradually. Finally, this system will end up with a dead end.<br> | ||
Revision as of 07:32, 5 October 2011
Introduction
According to the five clear quorum sensing system we summarize in the New QS Device, we could build a promoting cycle imitating the Wu xing’s promoting cycle.
General mechanism
In the promoting cycle, Every device of our five element cycle is composed by 4 basic segments: activator protein production; AHL synthetase production; protein LeuB production and reporter fluorescent protein expression.
The first promoter is ptetR，as the E.coli chassis we used do not contains the tetR, so it have a constitutive expression, generating the activator protein, like luxR，lasR, cinR, sinR, rhiR. When the input signal, namely AHLs, meet the activator. They will bind with each other and dimerize, forming a dimmer, which will bind the cassette in the second promoter in the picture, activating the downstream genes’ expression.
So the signal generator, like lasI，cinI，sinI，rhiI，luxI，will produces their specific AHLs, and the leuB is produced, making the leucine deficient HB101 be able to live in a medium without leucine.
When the reporter fluorescent protein is detected under the fluorescence microscope, we could know that this circuit has worked.
As for the specific promoting circuit, we could see them as below.
The specific mechanism in each device
First, let’s take the device I as an example.
The upper half circle is a promoting circuit, we will only discuss the upper half one here, for the bottom half one, you can see them in theRestraining cycle Restraining cycle .
The activator protein luxR in device 1 is generated constitutively. When the signal molecule 3-O-C6-HSL is added in the medium, the luxR will bind with the 3-O-C6-HSL (produced by luxI in device 5) to activate the promoter--plux. When plux is activated, another AHL synthetase lasI will express, producing the signal molecule 3-O-C12-HSL, which can be released out of the cell and be received by the E.coli with device 2.
We use strain HB101 whose leuB gene was mutated invalid in genome, thus disabling its ability to produce LeuB by itself. The expression of leuB in promoting circuit will make up for the deficiency and is necessary for the survival of bacteria on medium without leucine. Finally, the reporter proteins EYFP will tell us whether the device works or not.
Through the same mechanism, strain with device 2 receives molecule 3-O-C12-HSL( released by device 1), activates lasR, then produces cinI, catalyze the formation of 3-OH-C14:1-HSL, produces leuB for self-survival, a different color fluorescent protein generates. It is the same with the other 3 devices left.
The promoting cycle constituted by five devices
he final results we want to achieve
When and only when the five kinds of bacteria were cultured together in complementary medium without leucine, they will survive and showing five different fluorescent proteins when detected by light with different wave lengths. Once any kind of bacteria is dead or lacked in the culture, the whole cycle will break off as a result of the AHLs ‘ signal blocking . correspondingly, AHL synthetase, LeuB and reporter protein’s expression will decreased gradually. Finally, this system will end up with a dead end.
All in all, every device relys on each other tightly , one support another and form a promoting cycle. Their mutual relationship can be summed up into one word.
